北京油鸡肠道肽转运蛋白PEPT1表达载体的构建及在293-T细胞的表达

Cloning and constructing recombinant Beijing-you chicken PEPT1 in 293-T cell

本试验通过分子生物学手段建立了北京油鸡PEPT1细胞模型。根据GenBank的原鸡PEPT1保守区序列设计引物,从北京油鸡肠道粘膜组织中扩增出PEPT1基因,将其与pGEM-T载体连接并测定核苷酸序列,成功获得到测序正确的2145bp的基因,并将北京油鸡肠肽转运载体PEPT1基因克隆到真核表达载体pcDNA3.0,构建了真核表达载体pcDNA3-PEPT1。采用脂质体介导将表达质粒pcDNA3-PEPT1转染293-T细胞,同时转染pcDNA3.0-EGFP荧光蛋白进行转染体系荧光监测,流式细胞术检测转录后16、20、24h的荧光强度。分别在16、20、24、44h收集等量转染细胞,抽提转染细胞总RNA,DNAseⅠ处理残留的DNA污染,反转录合成cDNA,以构建不同稀释度的pGEM-T-PEPT1质粒为模板,建立SYBERGREEN实时荧光定量标准曲线,检测PEPT1在293-T细胞中的转录水平。结果表明,在质粒转染入293-T细胞后,在16、20、24、44h均有稳定的转录水平。从而建立了在293-T细胞表达北京油鸡PEPT1的外源模型,同时为研究该转运蛋白性质,进一步调控动物肠道肽的吸收奠定了基础。

This study is to establish Beijing-you chicken PEPT1 cell model. According to the PEPT1 gene sequence in GenBank, a pair of specific sequences as primers was designed to amplify the PEPT1 sequence isolated from intestine of Beijing-you chicken jejunum cell by RT-PCR, the PEPT1 gene was amplified successfully and the PCR products were cloned into pGEM-T vector. Sequence analysis indicated that the length of the PEPT1 gene was 2 145 bp and its sequence was correct. Then this gene was insert into expression vector pcDNA3.0. The constructed pcDNA3.0-PEPT1 was expressed in 293-T cells with pcDNA3.0-EGFP plasmid by lipofectin methods. Fluorescence level was detected by flow cytometer in 16, 20, 24 h to detect positive ratio and extracted total RNA of the same cell, wipe out contaminated DNA by DNAse Ⅰ and get cDNA by reverse transcription, a pair of specific sequences as primers was to verifiy the PEPT1 expression level, use plasmid pGEM-T-PEPT1 to construct standard curve, by Real-Time PCR survey we found that there were steady transcription level in 16, 20, 24 and 44 h after pcDNA3.0-PEPT1 transfection. It was concluded that Beijing-you chicken PEPT1 can be expressed efficiently in eukaryotic expression vector pcDNA3.0-PEPT1 transfected 293-T cells. We lay the base for Beijing-you chicken PEPT1 cell model and future study to intestinal PEPT1 transportation in chicken.